Apoptosis Of Hematopoietic Stem Cells Research Articles

Distinct Roles of CHK2 and Cell Death in Suppressing Oncogenic Potential of Tandem DNA Breaks in Human Hematopoietic Stem Cells

DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells.Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing.Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC.To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress. DisclosuresNo relevant conflicts of interest to declare.

Slower Engraftment in Patients with High Expression of miRNA-15a, miRNA-16, miRNA-126, miRNA-146a, miRNA-223 Prior to Autologous Stem Cell Transplantation and at Early Time after Transplantation

IntroductionMicroRNAs are a class of small (19-25 nucleotides), endogenous RNA which play a significant role in regulation of gene expressions, cell proliferation, differentiation, apoptosis. These class of small molecules, engaged in angiogenesis and hematopoiesis can influence hematopoietic stem cells (HSC) homing after transplantation by targeting bone marrow niche microenvironment. The aim of the study was to evaluate expression of miRNA-15a, miRNA-16 (engaged in angiogenesis, apoptosis, proliferation and differentiation of HSC) miRNA-126, miRNA-146a (regulating homing of HSC after transplantion) and miRNA-223 (involved in apoptosis and granulopoiesis) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem transplantation (HSCT) and in early posttransplant period.PatientsFifty one patients were enrolled to the study (23 F, 28 M). The median (Me) age was 59 years. The investigated group consisted of 42 multiple myeloma (7 CR, 27 VGPR, 8 PR), 7 non-Hodgkin lymphoma (3 CR, 4 PR), and 2 Hodgkin lymphoma (2 PR). The blood (plasma samples) were collected on 4 time points: before chemotherapy - "BC", on the day of HSCT (after chemotherapy) - "0", seven days after HSCT "N" (nadir) - "N" and fourteen days after HSCT - "+14".MethodsTotal RNA was isolated using miRNeasy Serum/Plasma KIT (Qiagen, Hilden, Germany). MiRNA conversion to cDNA was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). We used TaqMan MicroRNA Assays (Applied Biosystems): has-miR-15a-5p, has-miR-15a-5p, has-miR-146a-5p, has-miR-126-3p and has-miR-223-3p primers) to detect mature miRNA. Reverse transcription conditions were normalized using 5 nmol mirVana miRNA Mimic (cel-miR-39) as an endogenous control (Ambion, Austin, TX). The miRNA level was evaluated by Real Time PCR method.The Wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: miRNA’s RQ levels at two different time points. Spearman rank correlation coefficient (R) was used to compare independent variables: regeneration and miRNA’s RQ level. Comparisons and correlations were considered significant if p<0.05.ResultsKinetics of miRNA before conditioning treatment and after HSCTOur research revealed significant, steady decline of miRNA-15a, miRNA-16, miRNA-126, miRNA-146a and miRNA-223 at all time points as compared to baseline (BC). We observed increased miRNA-126 and miRNA-223 expression on "+14" day as compared to "N" day (Tab. 1).Correlation between miRNA expression and regenerationWe observed that higher level of investigated miRNAs after chemotherapy resulted in longer time to engraftment. Our research revealed positive correlation between miRNA-15a, miRNA-16, miRNA-126 and miRNA146a levels on "0' day and the time to engraftment (R=0.36, p=0.009), (R=0.41, p=0.003), (R=0.30, p=0.03), (R=0.29, p=0.03) respectively. Additionally we found positive correlation between levels of miRNA-15a, miRNA-146a and miRNA-223 assessed on "N" day and time of regeneration (R=0.33, p=0.02), (R=0.34, p=0.02) and (R=0.30, p=0.04) respectively. No correlation between expression of all investigated miRNAs before chemotherapy and time to engraftment was found.ConclusionsIn conclusion, all investigated miRNAs changed significantly after conditioning treatment. Low expression of miRNA-15a and miRNA-16, as HSC proliferation and angiogenesis factors, play important role in regeneration process after chemotherapy and in early post-transplant period.Low expression of miRNA-223 on nadir day correlates with faster engraftment, which is probably related with miRNA-223 activity on different stages of hematopoietic cells development, especially myeloid precursors differentiation and maturation. Effective regeneration, correlated with decreased miRNA-126, miRNA-146a level after conditioning treatment, may be affected by well-known homing processes regulated by VCAM-1 and CXCR4/SDF-1 signaling. We suggest that investigated miRNAs expression in the early post-transplant period is significant for time to engraftment after HSCT. [Display omitted] DisclosuresGrzybowska-Izydorczyk:BMS: Honoraria; Novartis: Honoraria. Robak:AbbVie: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p&lt;0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p&lt;0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p&lt;0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by &gt;2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p&lt;0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

Low Doses of Oxygen Ion Irradiation Cause Acute Damage to Hematopoietic Cells in Mice.

One of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.

282. Circumventing Hematopoietic Toxicities and Reversing Sickle Phenotype by Lineage-Specific BCL11A Knock Down and γ-Globin Induction

The fetal hemoglobin repressor BCL11A represents a therapeutic target for β-hemoglobinopathies as reduced expression of BCL11A leads to simultaneous increased γ-globin and reduced β-globin expression. Reversing this hemoglobin switch is particularly relevant in sickle cell disease to reduce the βS concentration and increase expression of the protective fetal hemoglobin (HbF, α2γ2). Here we show that despite use of optimized shRNAs embedded into a miRNA (shRNAmiR) architecture to reduce non-specific cellular toxicities (Guda et al. MT, 2015), the knockdown of BCL11A profoundly and specifically impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs). In competitive transplantation assays cells transduced with shRNAmiRs targeting the BCL11A mRNA were consistently underrepresented after hematopoietic reconstitution with gene modified HSCs. Although this effect was particularly pronounced in the B-cell compartment, it was also the case for all other assessed hematopoietic lineages. Mechanistically, while knock-down of BCL11A did not lead to a detectable phenotype in terms of apoptosis, growth or differentiation in human or mouse HSCs in vitro, a significant increase in S/G2-phase human HSCs after engraftment into NSG mice was found, a phenotype associated with stem cell exhaustion. To avoid this BCL11A-specific HSC toxicity, we suppressed BCL11A in erythroid cells in a lineage-specific fashion by using transcriptional regulatory elements derived from the β-globin locus. Utilizing this approach for the expression of the optimized shRNAmiRs led to stable long-term engraftment of mouse and human gene modified cells in congenic or NSG-mice, respectively. Transduced primary normal or sickle cell disease (SCD) human HSCs gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60-70% γ-chain expression and a corresponding increase in HbF at low vector copy numbers per cell (VCN<1.5). Similar results were obtained after in vitro differentiation of CD34+ cells harvested 16 weeks following engraftment in NSG mice. Transplantation of gene modified murine HSCs from BERK sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key phenotypes of SCD. In summary, we have shown an unexpected and severe toxicity of BCL11A knockdown in repopulating HSCs that has direct and important consequences for translation into human gene therapy trials. By utilizing erythroid lineage-specific and microRNA embedded expression of targeting shRNAs we demonstrate the capacity of lentivirus vectors to effect significant γ-globin induction leading to clinically relevant increases in HbF while obviating HSC toxicity.

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.

Caught in a Wnt storm: Complexities of Wnt signaling in hematopoiesis.

The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.

Differential ability of MSCs isolated from placenta and cord as feeders for supporting ex vivo expansion of umbilical cord blood derived CD34(+) cells.

IntroductionEx vivo expansion of umbilical cord blood (UCB) is attempted to increase cell numbers to overcome the limitation of cell dose. Presently, suspension cultures or feeder mediated co-cultures are performed for expansion of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) have proved to be efficient feeders for the maintenance of HSCs. Here, we have established MSCs-HSCs co-culture system with MSCs isolated from less invasive and ethically acceptable sources like umbilical cord tissue (C-MSCs) and placenta (P-MSCs). MSCs derived from these tissues are often compared with bone marrow derived MSCs (BM-MSCs) which are considered as a gold standard. However, so far none of the studies have directly compared C-MSCs with P-MSCs as feeders for ex vivo expansion of HSCs. Thus, we for the first time performed a systematic comparison of hematopoietic supportive capability of C and P-MSCs using paired samples.MethodsUCB-derived CD34+ cells were isolated and co-cultured on irradiated C and P-MSCs for 10 days. C-MSCs and P-MSCs were isolated from the same donor. The cultures comprised of serum-free medium supplemented with 25 ng/ml each of SCF, TPO, Flt-3 L and IL-6. After 10 days cells were collected and analyzed for phenotype and functionality.ResultsC-MSCs and P-MSCs were found to be morphologically and phenotypically similar but exhibited differential ability to support ex vivo hematopoiesis. Cells expanded on P-MSCs showed higher percentage of primitive cells (CD34+CD38−), CFU (Colony forming unit) content and LTC-IC (Long term culture initiating cells) ability. CD34+ cells expanded on P-MSCs also exhibited better in vitro adhesion to fibronectin and migration towards SDF-1α and enhanced NOD/SCID repopulation ability, as compared to those grown on C-MSCs. P-MSCs were found to be closer to BM-MSCs in their ability to expand HSCs. P-MSCs supported expansion of functionally superior HSCs by virtue of reduction in apoptosis of primitive HSCs, higher Wnt and Notch activity, HGF secretion and cell-cell contact. On the other hand, C-MSCs facilitated expansion of progenitors (CD34+CD38+) and differentiated (CD34−CD38+) cells by secretion of IL1-α, β, MCP-2, 3 and MIP-3α.ConclusionsP-MSCs were found to be better feeders for ex vivo maintenance of primitive HSCs with higher engraftment potential than the cells expanded with C-MSCs as feeders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0194-y) contains supplementary material, which is available to authorized users.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes.

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Circulating microRNA as Biomarkers in Hematological Malignancies.

Hematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate differentiation, proliferation, and apoptosis of hematopoietic stem cells. Aberrant microRNA (miRNA) expression could affect normal hematopoiesis, leading to the development of hematological malignancies. Hematologic cancers, which are caused by malignant transformation of cells of the bone marrow and the lymphatic system, are usually divided into three major groups: leukemias, lymphomas, and monoclonal gammopathies. Hematologic malignancies are highly aggressive diseases with high morbidity and mortality. For these reasons, early and easily obtainable markers for diagnosis, risk stratification, and follow-up are essential for improvement of outcome and survival of these patients. Recent studies have provided new insights about the diagnostic value of expression patterns of miRNAs in serum/plasma in these diseases. While the use of circulating miRNAs is only at the experimental level, it appears to have a great potential. This chapter deals with the use of circulating miRNAs as minimally invasive biomarkers in hematologic malignancies.

The possible role of liver kinase B1 in hydroquinone-induced toxicity of murine fetal liver and bone marrow hematopoietic stem cells.

Epidemiological studies suggest that the increasing incidence of childhood leukemia may be due to maternal exposure to benzene, which is a known human carcinogen; however, the mechanisms involved remain unknown. Liver Kinase B1 (LKB1) acts as a regulator of cellular energy metabolism and functions to regulate hematopoietic stem cell (HSC) homeostasis. We hypothesize that LKB1 contributes to the deregulation of fetal or bone hematopoiesis caused by the benzene metabolite hydroquinone (HQ). To evaluate this hypothesis, we compared the effects of HQ on murine fetal liver hematopoietic stem cells (FL-HSCs) and bone marrow hematopoietic stem cells (BM-HSCs). FL-HSCs and BM-HSCs were isolated and enriched by a magnetic cell sorting system and exposed to various concentrations of HQ (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. We found that the inhibition of differentiation and growth, as well as the apoptosis rate of FL-HSCs, induced by HQ were consistent with the changes in BM-HSCs. Furthermore, G1 cell cycle arrest was observed in BM-HSCs and FL-HSCs in response to HQ. Importantly, FL-HSCs were more sensitive than BM-HSCs after exposure to HQ. The highest induction of LKB1 and adenosine monophosphate-activated protein kinase (AMPK) was observed with a much lower concentration of HQ in FL-HSCs than in BM-HSCs. LKB1 may play a critical role in apoptosis and cell cycle arrest of HQ-treated HSCs. This research has developed innovative ideas concerning benzene-induced hematopoietic toxicity or embryotoxicity, which can provide a new experimental evidence for preventing childhood leukemia. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 830-841, 2016.

Roles of NLRP1 in blood diseases

The inflammasome is a group of multiprotein complexes in the cytoplasm, which can activate caspase-1 that mediates the maturation and release of IL-1β, IL-18, IL-33 and other pro-inflammatory cytokines.NALP1 (NACHT leucine-rich-repeat protein 1), also known as NLRP1, is the first one of the identified complex inflammasomes with definite ligands mainly involved in the activation of inflammasome assembly and the formation of apoptotic bodies. Moreover, it was also found that NLRP1 plays an important biological role in the development of acute leukemia, the bone marrow hematopoietic stem cell apoptosis and other blood diseases. This review briefly summarizes the structure, activation mechanism, regulation and the role of NLRP1 in the hematopoietic system.

Challenges and opportunities of microRNAs in lymphomas.

MicroRNAs (miRNAs) are small non-coding RNAs that control the expression of many target messenger RNAs (mRNAs) involved in normal cell functions (differentiation, proliferation and apoptosis). Consequently their aberrant expression and/or functions are related to pathogenesis of many human diseases including cancers. Haematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate commitment, differentiation, proliferation, and apoptosis of hematopoietic stem cells (HSC). Alterations on this network could affect the normal haematopoiesis, leading to the development of haematological malignancies such as lymphomas. The incidence of lymphomas is rising and a significant proportion of patients are refractory to standard therapies. Accurate diagnosis, prognosis and therapy still require additional markers to be used for diagnostic and prognostic purpose and evaluation of clinical outcome. The dysregulated expression or function of miRNAs in various types of lymphomas has been associated with lymphoma pathogenesis. Indeed, many recent findings suggest that almost all lymphomas seem to have a distinct and specific miRNA profile and some miRNAs are related to therapy resistance or have a distinct kinetics during therapy. MiRNAs are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum where they are highly stable and quantifiable within the diagnostic laboratory at each consultation. Accordingly they could be specific biomarkers for lymphoma diagnosis, as well as useful for evaluating prognosis or disease response to the therapy, especially for evaluation of early relapse detection and for greatly assisting clinical decisions making. Here we summarize the current knowledge on the role of miRNAs in normal and aberrant lymphopoiesis in order to highlight their clinical value as specific diagnosis and prognosis markers of lymphoid malignancies or for prediction of therapy response. Finally, we discuss their controversial therapeutic role and future applications in therapy by modulating miRNA.

Protective Effects of Tannins in Sanguisorbae Radix on Myelosuppression Mice

ObjectiveTo explore the protective effects of tannins in Sanguisorba Radix (TSR) on myelosuppression mice induced by cyclophosphamide (CTX). MethodsTSR was ig given at the dose of 20 mg/kg for 10 d after ip administration of CTX (200 mg/kg). ResultsTSR could significantly increase the numbers of white blood cells, red blood cells, and platelets of myelosuppression in mice. And it could accelerate bone marrow haemopoietic stem/progenitor cells (HSPCs) in myelosuppression mice and enhance cell proliferation by promoting cell cycles from G0/G1 phase to access into S and G2/M phases, then the reduced number of HSPCs induced by CTX was reversed. Moreover, TSR could increase the mRNA and protein expression levels of O(6)-methylguanine-DNA methyltransferase (MGMT) in HSPCs of myelosuppression mice. ConclsionTSR has a protective function against CTX-induced myelosuppression. The mechanism might be related to protecting hematopoietic stem cells of bone marrow, stimulating hematopoiesis recovery, as well as preventing the apoptosis of hematopoietic stem cells induced by CTX.

Selenium suppresses leukemia through the action of endogenous eicosanoids.

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ(12)-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia.

Knockdown of Puma protects cord blood CD34(+) cells against γ- irradiation

Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

Hematopoietic Stem Cell Injury Induced by Ionizing Radiation

Exposure to ionizing radiation (IR) as the result of nuclear accidents or terrorist attacks is a significant threat and a major medical concern. Hematopoietic stem cell (HSC) injury is the primary cause of death after accidental or intentional exposure to a moderate or high dose of IR. Protecting HSCs from IR should be a primary goal in the development of novel medical countermeasures against radiation. Significant progress has been made in our understanding of the mechanisms by which IR causes HSC damage. The mechanisms include (i) induction of HSC apoptosis via the p53-Puma pathway; (ii) promotion of HSC differentiation via the activation of the G-CSF/Stat3/BATF-dependent differentiation checkpoint; (iii) induction of HSC senescence via the ROS-p38 pathway; and (iv) damage to the HSC niche. Induction of apoptosis in HSCs and hematopoietic progenitor cells is primarily responsible for IR-induced acute bone marrow (BM) injury. Long-term BM suppression caused by IR is mainly attributable to the induction of HSC senescence. However, the promotion of HSC differentiation and damage to the HSC niche can contribute to both the acute and long-term effects of IR on the hematopoietic system. In this review, we have summarized a number of recent findings that provide new insights into the mechanisms whereby IR damages HSCs. These findings will provide new opportunities for developing a mechanism-based strategy to prevent and/or mitigate IR-induced BM suppression. Antioxid.

Effect of PD0332991 on biological activity of hematopoietic stem cells in mice

To investigate the effect of PD0332991 (C24H29N7O2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice. The self renewal ability of HSCs was measured by cobblestone forming cell assay (CAFC). The colony-forming cell (CFC) assay was used to quantify the changes of numbers and functions of HPC after the treatment of the compound. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were analyzed by flow cytometry. There were obvious changes in cell cycle regulation between control and PD0332991 groups. HSCs in G1 phase increased significantly from 76.3% to 89.5% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 20.5% to 7.3% (P<0.05). HPCs in G1 phase also increased from 74% to 87.4% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 25.54% to 11.6% (P<0.05). The apoptotic fractions between control and PD0332991 groups had no statistical difference (P>0.05). After cultured with PD0332991, the expression levels of cell cycle genes CDK1, CyclinA2, CyclinF, p18, p19 and p27 decreased by 58.77%, 66.35%, 56.33%, 62.18%, 32.28% and 36.53% respectively, while expression of CDK7 increased by 27.27% (P<0.05). No visible expression difference was observed in apoptosis and self-renew related genes. After treatment of PD0332991, the self-renewal ability of HSC decreased significantly. There were almost no CFCs in PD0332991 group in CAFC assay. Similarly, the frequency of CFCs was dramatically lower in PD0332991 group. These results suggested that PD0332991 affected HSC/HPC from mice mainly through inhibiting the cell cycle rather than apoptosis. It also suggested that CDK4/6 might play a key role in the regulation of HSC/HPC.

The TIF1β-HP1 System Maintains Transcriptional Integrity of Hematopoietic Stem Cells

SummaryTIF1β is a transcriptional corepressor that recruits repressive chromatin modifiers to target genes. Its biological function and physiological targets in somatic stem cells remain largely unknown. Here, we show that TIF1β is essential for the maintenance of hematopoietic stem cells (HSCs). Deletion of Tif1b in mice induced active cycling and apoptosis of HSCs and promoted egression of HSCs from the bone marrow, leading to rapid depletion of HSCs. Strikingly, Tif1b-deficient HSCs showed a strong trend of ectopic expression of nonhematopoietic genes. Levels of heterochromatin protein 1 (HP1α, β and γ) proteins, which form a complex with TIF1β, were significantly reduced in the absence of TIF1β and depletion of HP1 recapitulated a part of the phenotypes of Tif1b-deficient HSCs. These results demonstrate that the TIF1β-HP1 system functions as a critical repressive machinery that targets genes not normally activated in the hematopoietic compartment, thereby maintaining the transcriptional signature specific to HSCs.

Flow cytometric analysis of signaling and apoptosis in hematopoietic stem cells.

The analysis of protein phosphorylation in hematopoietic stem cells (HSCs) provides a powerful tool for studying the cell signaling activities that mediate HSC fate decisions, such as self-renewal, differentiation, and apoptosis. The first part of this chapter describes a method of intracellular staining for phosphorylated proteins in conjunction with membrane staining for multiple hematopoietic cell-surface markers, and subsequent flow cytometric analysis of protein phosphorylation levels [indicated by mean fluorescence intensity (MFI) of specific fluorochromed phospho-antibodies] in primitive hematopoietic cells. The second part describes a method for assessing the frequency of apoptosis in HSCs using extracellular staining with recombinant Annexin V and 7-Amino-Actinomycin (7-AAD). Both parts involve an initial magnetic enrichment of hematopoietic stem/progenitor cells from bone marrow. Because of the intracellular detection required for the HSC signaling assay, this assay also includes cell fixation and permeabilization. Gating strategies for assessing MFI and the frequency of Annexin V(+) apoptotic cells in a complex population are also described along with representative examples.